Study design
The CoLaus (Cohorte Lausannoise) study is a population-based prospective study assessing the clinical, biological, and genetic determinants of cardiovascular disease aged 35 to 75 years at baseline, living in the city of Lausanne, Switzerland)13. In each survey, participants answered questionnaires, underwent a clinical examination and blood samples were drawn for analyses13. Recruitment began in June 2003 and ended in May 2006. For this cross-sectional analysis, all participants at were eligible and no control group was created.
Anthropometry
Anthropometric measurements were conducted using a standard methodology. Body weight and height were measured with participants barefoot and in light indoor clothes. Body weight was measured in kilograms to the nearest 100 g using a Seca® scale (Hamburg, Germany). Height was measured to the nearest 5 mm using a Seca® (Hamburg, Germany) height gauge14. Body mass index (BMI) was computed and categorized into underweight (< 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2) and obesity (≥ 30 kg/m2)15.
Waist circumference (WC) was measured mid-way between the lowest rib and the iliac crest, and hip was measured at the largest location, using a non-stretchable tape; the average of two measurements was taken14. Abdominal obesity was defined as a waist circumference > 102 cm (men) or > 88 cm (women). A high waist to height ratio (WHtR) was defined as > 0.516; the WHtR is considered as a good indicator of abdominal obesity17.
Fat and fat-free mass (in percent of total body weight) were assessed by electrical bioimpedance in the lying position after a 5-min rest using the Bodystat® 1500 body mass analyzer (Bodystat Ltd, Isle of Man, England)18. This device has been shown to correlate well (r = 0.968) with measurements from dual energy X-ray absorptiometry (DEXA)18.
The conicity index (CI) was calculated according to Valdez9. It is based upon the idea that people accumulate fat around the waist and, the shape of their bodies seems to change from that of a cylinder to that of a “double cone” (two cones with a common base). The CI is determined by the formula
$$CI=frac{Waist}{0.109times sqrt{frac{Body, weight}{Height}}}$$
The CI was further categorized as normal if < 1.25 and < 1.18 for men and women, respectively, and as high if ≥ 1.25 and ≥ 1.18 for men and women, respectively19.
Body roundness index (BRI)20 was computed according to and is based on waist circumference and height.
$$BRI=364.2-365.5times sqrt{1-left[frac{{left(frac{Waist}{2pi }right)}^{2}}{{left(0.5times Heightright)}^{2}}right]}$$
A Body shape index (ABSI)21 was computed according to and is based on waist, BMI and height. Since there are no clinical cutoff values for BRI or ABSI, high BRI and ABSI were defined as those within the highest quartile group (Q4).
$$ABSI=frac{Waist}{{BMI}^{2/3} times {Height}^{1/2}}$$
Vitamin D levels
Vitamin D was assessed at baseline through an ultra-HPLC tandem-MS system. The calibrators, 3Plus1 Multilevel Serum Calibrator Set 25-OH-Vitamin D3/D2 (ChromoSystems), were standardized against the National Institute of Standards and Technology 972 reference material. Serum 25(OH)D3 and 3-epi-25(OH)D3 were expressed in nanomoles per liter (conversion factor: 1 nmol/L = 0.4006 μg/L). The interday CV% was 4.6% at 40 nmol/L22. Vitamin D levels were further categorized as normal (≥ 30 ng/mL or ≥ 75 nmol/l), insufficiency (21 to 29 ng/mL or 50–75 nmol/l) and deficiency (< 20 ng/mL or < 50 nmol/l)23. Hypovitaminosis D was defined for vitamin D levels < 30 ng/mL or < 75 nmol/l, encompassing insufficiency plus deficiency24.
Other covariates
Educational level was categorized into university, high school, apprenticeship, and mandatory. Nationality as born in Switzerland or not. Smoking status was self-reported and categorized as never, former, and current. Physical activity was considered if the participant reported performing at least twice a week a minimum of 20 min of leisure-time physical activity13.
Adipokines (adiponectin and leptin) were assessed at baseline. Adiponectin was assessed by ELISA (R&D Systems, Inc, Minneapolis, USA), with a maximum inter-assay CV of 8.3% and a maximum intra-assay CV of 8.3%. Leptin was assessed by ELISA (American Laboratory Products Company, Windham, USA) with a maximum inter-assay CV of 12.8% and a maximum intra-assay CV of 5.8%. High sensitive C-reactive protein (CRP) was assessed by immunoassay and latex HS on a Modular P apparatus (Roche Diagnostics, Basel, Switzerland).
Exclusion criteria
Participants were excluded if they lacked any variable needed for the bivariate and the multivariate analyses. Hence, participants devoid of vitamin D data; without anthropometric measurements and any covariate needed for adjustment (education, smoking, BMI, or physical activity) were excluded. The exclusion procedure was conducted sequentially as follows: first, participants devoid of vitamin D data were excluded; of the remaining participants, those without anthropometric measurements were excluded; finally, of the remaining participants, those missing any covariate were excluded.
Statistical analysis
Statistical analyses were performed using Stata version 16.1 for Windows (Stata Corp, College Station, Texas, USA)25. Descriptive results were expressed as number of participants (percentage) for categorical variables and as average ± standard deviation or median [interquartile range] for continuous variables.
As adiposity measures differ between sexes, stratification on the latter was performed. The associations between vitamin D levels and anthropometric markers (BMI, waist, WHR, WHtR, %fat as assessed by bioimpedance, CI, BRI and AABSI) were assessed as follows. First, comparison of vitamin D levels according to categories of anthropometric markers was performed using one-way (bivariate) or multivariate analysis of variance. Multivariate analysis was adjusted for age (continuous), nationality (Swiss, other), month, smoking categories (never, former, current), vitamin D supplementation (yes, no) and physical activity (yes, no) and the results were expressed as adjusted mean ± standard error. Second, bivariate nonparametric Spearman correlations and their 95% CIs were calculated between vitamin D levels and anthropometric markers as continuous variables. A stepwise multivariate linear regression analysis with age (continuous), nationality (Swiss, other), month, smoking categories (never, former, current), vitamin D supplementation (yes, no) and physical activity (yes, no) as locked terms was conducted to identify the anthropometric marker most associated with vitamin D levels. For simplicity, all anthropometric markers were standardized (i.e., zero average and unit standard deviation) before the stepwise regression.
Third, the association between vitamin D deficiency and categories of anthropometric markers was assessed using chi-square (bivariate analysis) and multivariate logistic regression with vitamin D deficiency (yes, no) as the dependent variable and adjusting for age (continuous), nationality (Swiss, other), month, smoking categories (never, former, current), vitamin D supplementation (yes, no) and physical activity (yes, no).
Sensitivity analyses were conducted by excluding participants receiving medically prescribed vitamin D levels. Finally, the analysis of the associations between vitamin D levels and leptin and adiponectin levels as obesity markers was conducted using Spearman correlation and linear regression adjusting for the aforementioned covariates; results of the linear regression were expressed as standardized coefficients. Statistical significance was considered for a two-sided test with p < 0.05.
Ethical statement
The institutional Ethics Committee of the University of Lausanne, which afterwards became the Ethics Commission of Canton Vaud (https://www.cer-vd.ch) approved the baseline CoLaus study (reference 16/03, decisions of 13th January and 10th February 2003). The study was performed in agreement with the Helsinki declaration and its former amendments, and in accordance with the applicable Swiss legislation. All participants gave their signed informed consent before entering the study. Data analysis was conducted in Switzerland and no data was shared with outside groups.